While tremendous progress has been made in the treatment of pediatric cancers over the past several decades, this disease remains one of the leading causes of mortality in children. Additionally, current therapies are associated with significant side effects and long-term morbidity. Thus, new, less toxic therapies are needed to achieve greater cure rates while preserving future health. The long-term objective of this proposal is to understand how cells can be manipulated to induce efficient transdifferentiation, by first determining the cellular mechanisms responsible for lineage specification and the limitation of differentiation potentials. This will lead to a potentially new strategic paradigm fo cancer therapy invoking differentiation and growth arrest rather than cytotoxicity. The central hypothesis of this proposal is that lineage restriction results from lineage-determined chromatin organization and transcription factor binding patterns that are defined by precise genetic and epigenetic determinants. Preliminary studies using the bHLH factors MyoD and NeuroD2 as a model indicate that the binding of these differentiation factors to specific targets in the genome is a critical step in lineage-specific gene activation, and that the protection of these sites from these factors may represent a fundamental mechanism used for lineage specification and restriction. To test this hypothesis, the experiments described in this proposal will employ high resolution structural modeling, chromatin immunoprecipitation followed by high-throughput sequencing, and nuclease accessibility assays to investigate the molecular mechanisms used by cells to restrict their lineage potentials. The results of these studies will determine: (1) if binding to lineage specific sites is sufficient for activation of lineage specific genes, regardles of the identity of the transactivating factor; (2) if additional regulatory mechanisms and cooperative factors are required for lineage specific gene activation; (3) the extent to which transcription factor binding site accessibility, as defined by the chromatin state, determines a cell's differentiation potential. In addition, an siRNA screen will be used to identify cellular factors tat inhibit differentiation in response to MyoD and NeuroD2. These may represent potential therapeutic targets to allow for transdifferentiation of resistant cells. Together, these studies wll significantly advance our ability to induce cellular transdifferentiation, and will establish a rational paradigm for inducing terminal differentiation in cancer cells. My long-term goal is to establish a research-based career in academic Pediatric Hematology-Oncology focused on broadening the current ways of thinking about the origin of pediatric malignancies and developing new therapeutic strategies through basic science discovery that will improve outcomes for children with cancer. As a physician-scientist with positions at both Seattle Children's Hospital and Fred Hutchinson Cancer Research Center, I am in an ideal setting to address relevant clinical problems using scientifically rational and rigorous approaches. The combination of clinical work at a highly ranked and dedicated pediatric hospital and basic science research at a world-class research facility possessing state-of-the-art equipment and resources, will facilitate achievement of the aims of this proposal, as well as my long-term career goals. Specifically, I have planned to engage in collaborative science and receive mentorship from well-established researchers at FHCRC, and to actively participate in the wealth of seminars, meetings, courses, and core facilities to achieve both my scientific and clinical goals, as well as to augment my training. The immediate goals of this project are to advance our understanding of how restriction of cell fate occurs during the process of differentiation. This represents a critical step towards achieving the long-term goals of understanding how malignant transformation occurs within cells and developing new treatment strategies for pediatric cancers.